多重PCR／RT-PCR技术检测PRRSV、PPV、PRV和PCV-2  

Development of a multiplex PCR/RT-PCR assay for detection of porcine reproductive and respiratory syndrome virus,porcine parvovirus,pseudorabies virus and porcine circovirus type 2

作　　者：王娟萍[1] 姚敬明[1] 高英杰[2] 金扩世[3] 丁馥香[1] 周胜花[1] 曹日亮[1] 贺东昌[1] 雷宇平 

机构地区：[1]山西省农业科学院畜牧兽医研究所,山西太原030032 [2]吉林大学畜牧兽医学院,吉林长春130062 [3]军事医学科学院军事兽医研究所,吉林长春130062 [4]山西省家畜疫病防治站,山西太原030024

出　　处：《中国兽医学报》2009年第3期258-262,共5页Chinese Journal of Veterinary Science

基　　金：山西省科技攻关项目(2006031056)

摘　　要：根椐GenBank中已发表的猪蓝耳病毒(PRRSV)、猪细小病毒(PPV)、伪狂犬病毒(PRV)和猪圆环病毒Ⅱ型(PCV-2)等4种病毒基因序列,对各病毒基因区进行同源性分析,确定PRRSV M和N、PPV VP2、PRVgD、PCV-2ORF2基因的保守区为各病毒的诊断靶序列。在建立各病毒单项PCR技术的基础上,优化多重PCR反应条件,建立了4种病毒的四重PCR技术,可同时扩增PRRSV的660 bp(北美株),PPV的313 bp,PRV的217 bp,PCV-2的447 bp的特异性片段。用82份临床病料对本研究多重PCR技术和单项PCR/RT-PCR技术进行对比验证,结果显示,两者的总符合率为93%以上。表明建立的多重PCR检测方法,具有特异、快速、准确的特点,可用于对这4种病毒的同时检测和鉴别诊断。Porcine reproductive and respiratory syndrome virus （PRRSV）, Porcine parvovirus （PPV）, Pseudorabies virus（PRV） and Porcine circovirus type 2 （PCV-2） are four main viral pathogens of porcine reproduction barrier diseases. To identify and differentiate rapidly the cause（s） of clinical diseases, a multiple PCR/RT-PCR assay was developed. Based on the similarity of the viral sequences deposited in the GenBank database, the M and N genes of PRRSV,the VP2 gene of PPV,the gD gene of PRV, and the ORF2 gene of PCV-2 were selected as the diagnostic targets. By using four pairs of virus-specific primers,four PCR/RT-PCR assay were ＂established to amplify the conservative regions of the four viruses, respectively. Consequently, a multiplex PCR/RT PCR method to detect the four viruses in one tube was developed. The multiple PCR/RT-PCR system would amplify a 660 bp fragment for PRRSV,a 313 bp for PPV,a 217 bp for PRV and a 447 bp for PCV-2 simultaneously or separately in the samples, depending on its infection status. To evaluate the multiplex PCR/RT-PCR assay,82 clinical samples were comparatively detected. The data showed that the multiple PCR method being 92% coincidence with the single PCRs, and it can be used for this four virus detection and differential diagnosis.

关 键 词：多重PCR PRRSV PPV PRV PCV2 

分 类 号：S852.65[农业科学—基础兽医学]